Analyte mass spectrometry quantitation using a universal reporter

ABSTRACT

Quantitation of analytes, including but not limited to peptides, polypeptides, and proteins, in mass spectrometry using a labeled peptide coupled to a reporter, and a universal reporter.

RELATED APPLICATIONS

This application is a National Phase of co-pending InternationalApplication No. PCT/US2011/043159, filed Jul. 7, 2011 which is acontinuation-in-part of International Application No. PCT/US2011/038629,filed May 31, 2011; each claiming priority to U.S. Patent ApplicationNo. 61/361,970, filed Jul. 7, 2010. the entirety of each is expresslyincorporated herein by reference in its entirety.

This application claims priority to co-pending PCT/US2011/038629 filedMay 31, 2011, and U.S. Application Ser. No. 61/361,970 filed Jul. 7,2010, each of which is expressly incorporated by reference herein in itsentirety.

Mass spectrometry (MS), in conjunction with internal standard peptideslabeled or modified with stable heavy isotopes resulting in a heavyisotope labeled sequence or, stated differently, a sequence modifiedwith a stable heavy isotope, provides fast, accurate and preciseabsolute quantitation of peptides, polypeptides, and proteins inbiological and other samples. This method is based on isotopic dilutionmass spectrometry (IDMS), also known as AQUA (WO 03/016861).

IDMS has limitations, as discussed below. Improvements such as theinventive method are thus desirable.

The inventive method resulted in absolute quantification of analytes byMS, and enabled a simple concentration calibration of analytes inreference solutions. The method used a heavy isotope labeled analyte(internal standard) that is in equimolar concentration with, and that iscleavably coupled to, a reporter R (that may or may not be heavy isotopelabeled); and a heavy isotope labeled universal reporter U. Analytesinclude, but are not limited to, peptides, polypeptides, and proteins,and may be RNAi, DNA, and lipids. Universal reporter U includes, but isnot limited to, peptides (i.e., polymers of amino acids) and otherpolymers.

In one embodiment the inventive method resulted in absolutequantification of peptide, polypeptide, and proteins analytes by MS. Themethod used a heavy isotope labeled peptide (proteotypic peptide,described below; internal standard) that was present in equimolarconcentration with, and was cleavably coupled at a proteolytic site to,an optionally heavy isotope labeled reporter peptide R; and a heavylabeled universal reporter peptide U analyzed by amino acid analysis.The heavy isotope labeled peptide need not undergo amino acid analysis.In one embodiment, several different proteotypic peptides from a singleprotein, linked to separate reporter peptides R, were analyzed. In oneembodiment, several different proteotypic peptides concatenated into onepolypeptide, linked to a single reporter peptide R, were analyzed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows peptides for mass spectrometry (MS) quantitation.

FIG. 2 shows a configuration for a peptide to be quantified linked to areporter peptide and correlated to a universal peptide U.

FIG. 3 shows an embodiment for quantitation of more than one peptide,each peptide linked to a separate reporter peptide R.

FIG. 4 shows an embodiment with three concatenated peptides linked to asingle reporter peptide R.

FIG. 5 shows an embodiment for simultaneous assay of more than oneanalyte in a sample using a single assay (multiplexing) where threeproteotypic peptides, each cleavably linked to its own reporter peptideR and correlated to a single universal reporter peptide U.

FIG. 6 shows configuration and relationship among components.

FIG. 7 shows configuration and relationship among peptide components.

FIG. 8 shows a naming convention.

FIG. 9 shows results of a dilution series of one universal reporterpeptide U using linear (FIG. 9A) and logarithmic (FIG. 9B) scales.

FIG. 10 shows proteolysis efficiency of an exemplary heavy isotopelabeled peptide.

FIG. 11 shows data for an exemplary heavy isotope labeled peptide.

FIG. 12 shows a spectrograph of isotopologues.

FIG. 13 shows results of various secondary ion mass (SIM) methods A,(FIG. 13A), B, (FIG. 13B), and C (FIG. 13C).

FIG. 14 schematically shows use of flanking amino acid sequences ingenerating a universal reporter R.

FIG. 15 schematically shows a reporter sequence R positioned in theN-terminal.

FIG. 16 shows data evidencing a single injection dilution curvegeneration by mass spectrometry.

FIG. 17 provides data from a dilution curve mixture.

FIG. 18 provides information on isomers used to generate a singleinjection dilution curve.

FIG. 19 shows composition of an isotopologue mixture used to generate asingle injection dilution curve.

FIG. 20 provides isomer internal standards for a single injectiondilution curve.

FIG. 21 illustrates quantitation using a heavy isotope labeledproteotypic peptide-reporter peptide R1.

FIG. 22 shows quantitation data using two different reporter peptides Rand two heavy isotope labeled proteotypic peptides for one protein usingthe same reporter peptide R.

FIG. 23 show one embodiment of sample preparation.

FIG. 24 lists selected sequences of heavy isotope labeled polypeptidesand reporters R.

FIG. 25 illustrates quantitation using concatenated heavy isotopelabeled proteotypic peptides A-B-C-reporter peptide R2.

FIG. 26A and FIG. 26B show calibration curve information and data.

FIG. 27 are graphs of FIG. 26 data showing linear response (FIG. 27A)and dynamic range (FIG. 27B).

FIG. 28 show quality control chromatography (FIG. 28A) and massspectrometry (FIG. 28B) data for one sequence.

FIG. 29 shows examples of limits of detection (LOD) and limits ofquantitation (LOQ) for selected peptides.

FIG. 30 shows quantitative analysis for selected peptides.

FIG. 31 shows quantitative analysis for selected peptides.

FIG. 32 shows an exemplary computer system.

FIG. 33 shows an exemplary computing environment.

FIG. 1 shows proteotypic peptides A, B, and C from protein orpolypeptide P. A proteotypic peptide is a signature peptide thatfragments into a defined or predictable ion series following MSdissociation to allow specific identification and quantitation of theparent protein, whether in a purified form or from a complex mixture. Ithas characteristics that render it readily quantified. A signaturepeptide is an unambiguous identifier of a specific protein. Any proteincontains an average of between 10 and 100 signature peptides. Anysignature peptide meets most of the following criteria: easily detectedby mass spectroscopy, predictably and stably eluted from a liquidchromatography (LC) column, enriched by reversed phase high performanceliquid chromatography (RP-HPLC), good ionization, good fragmentation. Apeptide that is readily quantified meets most of the following criteria:readily synthesized, ability to be highly purified (>97%), soluble in≦20% acetonitrile, low non-specific binding, oxidation resistant,post-synthesis modification resistant, and a hydrophobicity orhydrophobicity index ≧10 and ≦40. The hydrophobicity index is describedin Krokhin, Molecular and Cellular Proteomics 3 (2004) 908, which isexpressly incorporated herein by reference. A peptide having ahydrophobicity index less than 10 may not be reproducibly resolved byRP-HPLC. A peptide having a hydrophobicity index greater than 40 may notbe reproducibly eluted from a RP-HPLC column.

The inventive method uses an internal standard that is the heavy stableisotope (labeled or modified) form of the analyte to be quantified, alsoreferred to as a heavy analyte and shown in FIG. 6. In the embodimentusing a proteotypic peptide, the internal standard is the labeled formof the proteotypic peptide, also referred to as a heavy proteotypicpeptide, as shown in FIG. 7.

IDMS refers to the use of peptide modified with the heavy stable isotope(heavy isotope-labeled peptide) as internal standards to establish theconcentration versus MS response relationship and to perform absolutequantitation of peptide. The heavy isotope labeled peptide has identicalproperties as the unlabeled peptide, except that its mass is shifted bythe incorporated isotope(s). As a result of this mass shift, a knownquantity of the isotope-labeled peptide can be used as an internalstandard for peptide quantitation. The IDMS method results in targetedmass spectrometry (selected reaction monitoring (SRM)/multiple reactionmonitoring (MRM)) quantitation of peptides in complex samples ormixtures. SRM encompasses the MS acquisition setup to quantify a list oftarget proteins by the quantitation of specific fragment ions fromproteotypic peptides of these target proteins. Targeted assaydevelopment must be fast, have high throughput, be sensitive, specific,targeted, robust, reproducible, and cost effective; it typically usesliquid chromatography-tandem mass spectrometry (LC-MS/MS, LC/MS²).However, precise quantitation of large number of peptides in targetedproteomics experiments using SRM remains challenging.

In IDMS, the native proteotypic peptide differs from the heavyproteotypic peptide only due to insertion of a heavy amino acid. A heavyamino acid contains ¹³C (a heavy isotope of carbon) and/or ¹⁵N (a heavyisotope of nitrogen). The insertion of a heavy amino acid results inHeavyPeptide AQUA®, which differs from the proteotypic peptide only bythe difference in mass. The purity of the heavy peptide is increasedto >97% using preparative high performance liquid chromatography (HPLC).The precise quantity of HeavyPeptide AQUA® is determined by amino acidanalysis. The mixture of the peptide to be quantified and HeavyPeptideAQUA® as the internal standard yields two peaks in mass spectroscopy:the two peaks have the same elution time, but different masses.HeavyPeptide AQUA® is spiked into the sample to be analyzed at a knowquantity, making it possible to use its quantity to calculate thequantity of the peptide to be analyzed from the peak surfaces. Themethod compares the surface of the corresponding MS peak from the heavyisotope labeled peptide, with the peak of the non-labeled peptide withthe exact same sequence originating from the analyte (e.g., polymer,protein, peptide, or polypeptide) being quantified. The quantitationprecision is directly correlated to the accuracy of the quantity of theheavy peptide added to the sample.

The following example, while used specifically with a protein analyte,illustrates the general method applicable for analytes, whether proteinor non-protein. A sample (e.g., biological sample, food sample)containing numerous proteins is treated with a cleavage or digestionagent such as a protease (e.g., trypsin). Trypsin cleaves at each Ramino acid and K amino acid, yielding numerous fragments, each fragmenthaving about 13 amino acids (range 6 amino acids to 20 amino acids, upto 33 amino acids). Into this fragment-containing sample to be analyzedis introduced (spiked) one, two, or three HeavyPeptide AQUA® internalstandards, and quantitation is performed as described. In embodimentsusing proteolytic digestion, the quantitation precision is also directlycorrelated to the digestion predictability and efficiency.

In one embodiment, proteins contain one, two, or three proteotypicpeptide sequences, labeled as heavy or light (HeavyPeptide AQUA®,QuantPro®, or Ultimate®). The samples to be analyzed are spiked with theproteotypic peptides and quantitated by LC-MS/MS.

In IDMS, the internal standard has the same sequence as the proteotypicpeptide from the protein to be quantified, but the internal standard hasa different mass from the proteotypic peptide. The sequence of theinternal standard is thus pre-determined by the protein sequence; itcannot be changed The internal standard must be quantified by amino acidanalysis. Because the protein or polypeptide to be quantified differswith each experiment, the internal standard for this protein orpolypeptide necessarily also differs with each experiment, and requiresthat amino acid analysis be performed with each experiment. Eachquantitation requires a dilution curve that typically encompasses sixpoints, each point requiring about one hour of MS time, prior to actualsample quantitation. The costs for amino acid analysis are relativelyhigh and the procedure is time consuming. Each peptide sequence hasspecific solubility, and its non-specific binding constant varies basedupon various factors that may differ with each analysis, e.g., vesselmaterial, buffer, temperature, etc. Such variability decreases precisionand reproducibility.

In contrast, with peptides as a non-limiting example, the inventivemethod using a modified, optimized, labeled universal reporter U, andone analyte, more than one analyte, or several concatenated analytes,increased the analytical precision of SRM where quality of internalstandards is decisive to ensure precise quantification. Only thisuniversal reporter U undergoes amino acid analysis, rather than aninternal standard for each peptide to be quantified requiring amino acidanalysis. Universal reporter U quantification thus need be performedonly once, rather than with each experiment. The universal reporter Ucan be stocked and made readily available. In one embodiment, universalreporter U is labeled with a fluorophore, chemiluminescent molecule,and/or chromophore, and universal reporter U is quantified by measuringthe absorbance of the fluorophore and/or chromophore and/or intensity ofthe chemiluminescent molecule. For peptide analytes, no amino acidanalysis is required. In one embodiment, universal reporter peptide Ucontains one tryptophan, and universal reporter peptide U is quantifiedby measuring absorbance using the specific extinction factor of thetryptophan.

As shown in FIG. 2 using a peptide analyte, in one embodiment peptideA*, the internal standard, is linked with a reporter peptide R through acleavable site (e.g., proteolytic site) between A* and R. In thisembodiment, each of peptide A* and reporter peptide R contain at leastone amino acid labeled with a heavy isotope, known as a heavy aminoacid. When reporter peptide R is labeled with a heavy isotope, andbecause universal reporter peptide U must have a different mass,universal reporter peptide U can be represented with two heavy aminoacids, and reporter peptide R one heavy amino acid. However, there areother ways to obtain a difference in atomic mass; e.g., using differentheavy amino acids for reporter peptide R and universal reporter peptideU to obtain a difference in atomic mass.

Peptide A* has the same sequence as proteotypic peptide A, but peptideA* has a different mass due to the presence of the heavy amino acid.Universal reporter peptide U is a peptide standard for reporter peptideR. Universal reporter peptide U is not the internal standard used toquantify the protein or polypeptide. Universal reporter peptide U hasthe exact same sequence as reporter peptide R but has a different atomicmass.

In the ligation between peptide A* and reporter peptide R, resulting ina polypeptide, the reporter peptide R can be C-terminal to A*, i.e.,R-A*, or the reporter peptide R can be N-terminal to A*, i.e., A*-R. Thenomenclature A*-R is used to represent either the A*-R polypeptide orthe R-A* polypeptide. In either case when A* is a proteotypic peptide,there must be a cleavable (e.g., proteolytic) site between peptide A*and reporter peptide R in the resulting polypeptide.

The polypeptide A*-R is mixed with the sample that contains the proteinor polypeptide P to be quantified. A known quantity of universalreporter peptide U is added to the sample, i.e., universal reporterpeptide U is spiked into the sample. The sample is digested with aprotease (e.g. trypsin) that cleaves the polypeptide bonds. As a resultof protease action, polypeptide A*-R must be fully digested. In oneembodiment, universal reporter peptide U is added before cleavage (e.g.,proteolytic digestion). In one embodiment, universal reporter peptide Uis added after cleavage (e.g., proteolytic digestion).

After digestion the concentration of peptide A* and reporter peptide Rin the sample is equimolar. That is, the quantity of peptide A* is equalto the quantity of reporter peptide R. Universal reporter peptide U isused to quantify reporter peptide R using MS quantitation. The quantityof peptide A*, resulting from the proteolytic digestion of protein orpolypeptide P, is used to measure the quantity of peptide A in thesample.

In the embodiment using a peptide shown in FIG. 3, the same method isapplied to proteotypic peptides B and C from protein P in order toincrease the specificity of the quantitation. Peptide B* has the samesequence as proteotypic peptide B but has a different atomic mass due tothe presence of the heavy isotope labeled amino acid. Peptide C* has thesame sequence as proteotypic peptide C but has a different atomic massdue to the presence of the heavy isotope labeled amino acid.

Using a peptide embodiment as an example, A*-R includes a cleavage ordigestion site (e.g., proteolytic site) between A* and R. PolypeptideA*-R thus can be used as a pseudo-surrogate of protein or polypeptide Pto monitor proteolytic digestion in a single experiment, digestionefficiency among samples and experiments, and in some cases to normalizeresults from different samples and/or different experiments.

In examples using peptides, reporter peptide R can be optimized forproteolytic digestion. As one example, reporter peptide R can beselected and/or modified so that it contains a specific amino acid(e.g., tryptophan) that is easily quantified by absorption measurements.As shown in FIG. 5, reporter peptide R may contain more than one heavyisotope labeled amino acid. This embodiment increases the multiplexingpossibilities of the method by increasing the number of possible atomicmasses for the same reporter peptide R sequence, so that multiplepeptides can be quantified using universal reporter peptide U in asingle experiment.

In this multiplexing embodiment, reporter peptide R is synthesized withdifferent atomic masses, using standard methods known in the art. Asshown in FIG. 5, peptides A, B, and C from protein or polypeptide P arequantified in a single experiment using heavy peptides A*, B*, and C*,respectively, and using reporter peptides R1, R2, R3. In the embodimentshown in FIG. 5, reporter peptides R1, R2, R3, and universal reporterpeptide U, have the same sequence but different atomic masses. Tomaximize the number of mass combinations available for reporter peptideR, the sequence may be composed of, but is not limited to, one or moreof the following amino acids: alanine, arginine, isoleucine, leucine,lysine, phenylalanine, valine. These amino acids have a mass shift ordifference ≧4 Da. The minimum mass difference between the proteotypicpeptide (e.g., A), and the internal standard (e.g., A*), should exceedthe sensitivity threshold determination for MS differentiation. In oneembodiment, the minimum mass difference between the proteotypic peptideand the internal standard is 4 kDa when 4 kDa is the minimum atomic massdifference that can be discriminated. The number of peptides that can bequantified simultaneously using a universal heavy peptide U is limitedonly by the number of mass difference combinations available within thesequence.

In another example using peptides, reporter peptide R may be designedwith a low hydrophobicity index, which will increase the aqueoussolubility of the polypeptide A*-R where peptide A* has a hydrophobicityindex ≧40 or where peptide A* is poorly soluble. One example of areporter peptide R having a sequence that renders it highly soluble isPVVVPR (SEQ ID NO. 1); it has a hydrophobicity index of 13.45. Oneexample of a reporter peptide R having a sequence that renders it highlysoluble is SSAAPPPPPR (SEQ ID NO. 2) with a Krokhin hydrophobicityfactor of 7.57. In one example, each of reporter peptide R and universalreporter peptide U contains a chromophore and/or fluorophore used forquantification by absorbance measurement. In the embodiment where bothuniversal reporter peptide U and reporter peptide R include achromophore and/or fluorophore, universal reporter peptide U can bequantified by measuring the absorption of the chromophore and/orfluorophore, and not by amino acid analysis. The process of protein orpolypeptide quantification by absorbance is more robust than the processof amino acid analysis. Protein or polypeptide quantification byabsorbance is considered more precise than protein or polypeptidequantification by amino acid analysis. Examples of a chromophore orfluorophore and methods of assessing their absorbance are known in theart.

In the embodiment shown in FIG. 4, the polypeptide contains threeproteotypic peptides, each labeled with a heavy amino acid, concatenatedwith a single reporter peptide R also labeled with a heavy isotope aminoacid, resulting in C*-B*-A*-R. Using this polypeptide C*-B*-A*-Rguarantees equimolar quantities of each of peptides A*, B* and C* andthus decreases quantitation variability compared to quantitation usingindividual peptides. This embodiment increases the number of peptidesthat can be quantified with the same sequence as that of universalreporter peptide U.

In one embodiment, A*-R, or B*-A*-R, or C*-B*-A*-R can be cleaved beforebeing introduced into the sample to be quantified.

In embodiments using proteotypic peptides, peptides A*, B*, C*, andreporter peptide R can be randomly arranged, as long as they are linkedthrough a cleavage or digestion site (e.g., proteolytic site).

The polypeptide shown in FIG. 4 contains three heavy isotope labeledpeptides (A*, B*, and C*), corresponding to target peptides A, B, and C,linked to reporter peptide R, also containing a heavy isotope label.Other embodiments are possible where R does not contain a heavy isotopelabel. Other embodiments are possible that contain various numbers (n)of labeled peptides corresponding to one or more target peptides, joinedwith one or more reporter peptides. The range for n is governed by,e.g., manufacturing feasibility, solubility, etc. as know to one skilledin the art. In one embodiment, a value of n up to 99 is possible. In oneembodiment, a value of n up to 49 is possible. In one embodiment, n=4.In one embodiment, n=5. In one embodiment, n=6. In one embodiment, n=7.In one embodiment, n=8. In one embodiment, n=9. In one embodiment, n=10.In one embodiment, n=11. In one embodiment, n=12.

Universal reporter peptide U and reporter peptide R can be designed withdifferent sequences for multiplex quantitation. The number of massdifference combinations determined by a peptide sequence is limited.When the number of peptides to be quantified exceeds the maximum numberof mass difference combinations available for reporter peptide R, onecan use additional sequences of universal reporter peptide U: e.g., U¹,U², . . . U^(n) where n is limited only by the number of peptides thatcan be simultaneously quantified by an instrument. As one example, thepolypeptide A*-R may have the amino acid sequence TTVSKTETSQVAPA SEQ IDNO. 3, with peptide A* having the sequence TETSQVAPA SEQ ID NO. 4, andreporter peptide R having the sequence TTVSK SEQ ID NO. 5, as disclosedin WO/2003/046148.

Because the sequence of universal reporter peptide U is not restrictedor limited, and because universal reporter peptide U is a product thatcan be readily ordered, stocked, maintained, and inventoried, its useprovides flexibility to MS peptide quantitation. In one embodiment, thesequence of universal reporter peptide U can be customized to minimizenon-specific binding of the peptide, polypeptide, or protein to, e.g., avessel, tips, tubing, etc. by selecting a sequence with a lowhydrophobicity index, e.g., PVVVPR SEQ ID NO. 1 which has ahydrophobicity index of 13.45 or SSAAPPPPPR (SEQ ID NO. 2), which has ahydrophobicity index of 7.57. In one embodiment, the sequence ofuniversal reporter peptide U can be customized to maximize solubility ofthe polypeptide A*-R. For example, because universal reporter peptide Uis used, the polypeptide A*-R need not be quantified precisely prior toMS analysis. This results in shorter manufacturing time and lower costin producing polypeptide A*-R. Peptide A* is quantified at very lowconcentration, at which its solubility is guaranteed, resulting inenhanced precision and repeatability.

Because quantitation of peptide A* is performed on the same instrumentused for the quantitation of reporter peptide R and within the same MSprocedure, it always reflects the quantity added into the sample and isindependent of eventual alteration, degradation, and partial loss ofpolypeptide A*-R during sample preparation, fractionation, and liquidchromatography separation prior to MS quantitation. When using themethod described in WO 03/016861, A* is provided in a knownconcentration that is too high for use without dilution; thus, it istypically diluted 1000 to 10,000. If the sequence of A* is relativelyhydrophobic and prone to non-specific binding, as is the case forβ-amyloid peptides, a significant quantity of the standard will be lostduring dilution. This decreases the method's precision. Because thesequence of universal reporter peptide U can be designed and optimizedto decrease non-specific binding, the dilution of universal reporterpeptide U is not prone to significant non-specific binding. Universalreporter peptide U is included in the sample to be quantified, andquantitation of reporter peptide R is performed in the diluted sample,thus non-specific binding of the standard (e.g., β-amyloid peptide) willnot decrease the method's precision.

The polypeptide A*-R is a pseudo-surrogate of protein P and can be usedto monitor cleavage (e.g., proteolytic digestion). It can be used tocompare sample-to-sample, and/or experiment-to-experiment, digestionefficiency. It can be used to normalize results from sample-to-sample,and/or from experiment-to-experiment.

In one embodiment, the inventive method is adapted to MS quantitation ofanalytes, including but not limited to peptides, polypeptides, andproteins, using a proteotypic peptide that is coupled, through acleavable site, to a reporter peptide R or other moiety. This is shownschematically in FIG. 6 for any analyte, and in FIG. 7 for a peptideanalyte. The heavy proteotypic peptide contains the same amino acidsequence, but a different atomic mass, as the native proteotypicpeptide. The heavy proteotypic peptide is in equimolar concentrationwith the reporter peptide R. In one embodiment, reporter peptide R islabeled with a heavy isotope. In one embodiment, reporter peptide R isnot labeled with a heavy isotope. Universal reporter peptide U has thesame sequence as reporter peptide R. Universal reporter peptide U has adifferent mass than reporter peptide R because it contains a heavyisotope label. Only universal reporter peptide U is quantified. Aftercleavage (e.g., proteolytic digestion), the heavy proteotypic peptideand the reporter peptide R are released at equimolar concentration intothe sample. The quantity of the reporter peptide R is determined usingthe quantity of heavy universal reporter peptide U.

Universal reporter peptide U is sequence independent and is used as aquantitation standard and a cleavage or digestion standard. Universalreporter peptide U has a peptide sequence that is identical to reporterpeptide R but is independent from the protein to be assayed. Because thesequence of universal reporter peptide U and reporter peptide R isidentical, the atomic mass difference between universal reporter peptideU and reporter peptide R is obtained using a heavy labeled reporterpeptide R and a heavy labeled universal reporter peptide U. The atomicmass difference is obtained by using different heavy labels in reporterpeptide R and universal reporter peptide U, or by using an additionalheavy amino acid in reporter peptide R or universal reporter peptide U.Reporter peptide R may have a lower atomic mass or a higher atomic massthan universal reporter peptide U.

As represented in FIG. 8, a convenient convention for naming componentsis as follows: proteotypic peptides are named as letters, e.g., A, B, C;heavy isotope labeled proteotypic peptides are named as letters with anasterisk indicating a heavy isotope label, e.g., A*, B*, C*; R is areporter; U is a universal reporter; amino acid bearing the heavyisotope label indicated by either conventional amino acid one- orthree-letter naming in bold font, e.g., either R or Arg indicates theamino acid arginine with a heavy isotope label; one composition ofconcatenated peptides and universal reporter, commercially availableunder the trademark HeavyPeptide IGNIS™, is A*B*C*R.

In one embodiment, the sequence of universal reporter peptide U isoptimized and/or customized to be compatible with the properties of theproteotypic peptide by optimizing chromatographic ionization andfragmentation properties. As one example, universal reporter peptide Uis modified to enhance ionization and/or desolvation by introducingadditional charge or hydrophobic properties. As one example, universalreporter peptide U is modified to enhance fragmentation by introducingan aspartate-proline (DP) group that contains a highly scissile bondthat fragments in tandem MS at lower collisions energies than otherdipeptide linkages. As one example, universal reporter peptide U ismodified to have a similar retention time on liquid chromatography asthe proteotypic peptide by choosing a reporter peptide with a similarhydrophobicity factor to the proteotypic peptide. Thus, universalreporter peptide U can be optimized by design. For example, the numberof mass combinations for the identical peptide sequence can be optimizedto increase the multiplexing capacity, yielding up to 100 proteinscapable of being quantified in a single assay. Yet because the peptidesequences are identical, only one dilution curve is required to quantifyuniversal reporter peptide U. By increasing the number of identicalsequences with different masses, the number of proteins that can bequantified in a single experiment increases, without concomitantincrease in instrumentation use and resources.

In one embodiment, the universal reporter peptide U was optimized forlow specific binding, high solubility, high MS signal intensity, and/ordesired liquid chromatography retention time. In one embodiment, itspeptide sequence was modified to change its chromatographic retentionproperties; this is one example of internal modification. In oneembodiment, its structure was modified by attaching tags to change itschromatographic retention properties; this is one example of externalmodification. In one embodiment, its structure was modified by attachingtags that themselves had been modified to change its chromatographicretention properties; this is another example of external modification.

In one embodiment, a universal polymer is used, where polymer is broadlydefined as a joined group of monomers. The monomers either need not bepeptides, or need not be entirely peptides. In one embodiment,polysaccharides (i.e., glycan monomers) are used as universal polymers(U^(polymer)). A polysaccharide is a combination of two or moremonosaccharides linked by glycosidic bonds. Examples of polysaccharidesinclude starch, cellulose, and glycogen. Their structures and synthesisare know in the art. In one embodiment, deoxyribonucleic acid (DNA) orribonucleic acid (RNA) are used as universal polymers (U^(polymer)).Their structures and synthesis are known in the art. Methods to detectand quantify nucleotides are well established, e.g., PCR, quantitativePCR. Nucleotides attached to an analyte can by used as a uniqueidentifier (i.e., “barcode”) of the analyte and for quantitationpurposes using PCR, quantitative PCR, or isotopic dilution.

In one example, one of the following peptide sequences shown in thetable below, currently used as retention time calibrator peptides, wasused as universal reporter peptide U. These peptides exhibitedsufficient ionization and had defined elution properties. The followingtable below shows their sequence, hydrophobicity, and chromatographbehavior on a Hypersil Gold C₁₈ column.

SEQ Hydrophobicity Retention Peptide ID No. Factor Time (min) SSAAPPPPPR2 7.57 4.77 GISNEGQNASIK 6 15.50 6.62 HVLTSIGEK 7 15.52 7.22 DIPVPKPK 817.65 7.67 IGDYAGIK 9 19.15 8.18 TASEFDSAIAQDK 10 25.88 9.01SAAGAFGPELSR 11 25.24 9.41 ELGQSGVDTYLQTK 12 28.37 9.63 SFANQPLEVVYSK 1334.96 10.67 GLILVGGYGTR 14 32.18 10.79 GILFVGSGVSGGEEGAR 15 34.52 10.86LTILEELR 16 37.30 11.87 NGFILDGFPR 17 40.42 12.16 ELASGLSFPVGFK 18 41.1912.21 LSSEAPALFQFDLK 19 46.66 12.85

Hydrophobicity was determined using calculations done with algorithmsdescribed in Spicer. et al (2007). Sequence-specific retentioncalculator. A family of peptide retention time prediction algorithms inreversed-phase HPLC: applicability to various chromatographic conditionsand columns. Anal Chem. 79(22):8762-8.

In one embodiment, the heavy isotope label is incorporated in theC-terminal amino acid. For example, using the peptide SSAAPPPPPR SEQ IDNO. 2, this embodiment can be represented as SSAAPPPPPR*, where theterminal R contains the heavy isotope label.

In one embodiment, the heavy isotope is incorporated in the peptide at aposition other than the C-terminus. One or more of the following aminoacids may be labeled with a heavy isotope: alanine, arginine,isoleucine, leucine, lysine, phenylalanine, valine. These amino acidshave a mass shift or difference >4 Da. Additionally, multiple aminoacids within the peptide can be labeled, and the same amino acid may belabeled with different isotopes, such as ¹³C₆-arginine (R) and ¹³C₆¹⁵N₄-arginine which would introduce a 6 Da and 10 Da mass shift,respectively. For example, using the peptide SSAAPPPPPR SEQ ID NO. 2where * indicates the amino acid containing the position of the heavyisotope label, the following positions are possible: S*SAAPPPPPR,SS*AAPPPPPR, SSA*APPPPPR, SSAA*PPPPPR, SSAAP*PPPPR, SSAAPP*PPPR,SSAAPPP*PPR, SSAAPPPP*PR, SSAAPPPPP*R, SSAAPPPPPR*, S*SAAPPPPPR*,SS*AAPPPPPR*, SSA*APPPPPR*, SSAA*PPPPPR*, SSAAP*PPPPR*, SSAAPP*PPPR*,SSAAPPP*PPR*, SSAAPPPP*PR*, SSAAPPPPP*R*, SSAAPPPPPR** (with respect toSSAAPPPPPR**, double labeling permits higher multiplexing; thequantitation is performed at the MS/MS level using fragments from theparent ion). This embodiment, where the heavy peptide is located at aposition other than the C-terminus, permits higher multiplexing with thesame reporter sequence.

For multiplexed assays, custom peptides are combined together intocomplex targeted assays. Each custom peptide has a differentcorresponding universal reporter U that elutes similarly to the custompeptide. This permits many peptides to be easily multiplexed andquantified across an LC gradient without cross contamination. Forexample, a multiplex analysis array contains any number of differentuniversal peptides U having the same amino acid sequence, but adifferent atomic mass due to the presence of a heavy isotope, and anumber of reporter peptides R, each reporter peptide R cleavably linkedto a different isotopically labeled proteotypic peptide to be quantifiedin a sample. The universal peptides U have substantially similarchromatography retention time as the custom peptide. In one embodiment,the heavy isotope label in the universal reporter peptide U is moved todifferent amino acids. This embodiment permits higher multiplex arraysusing the same universal reporter peptide U amino acid sequence.

In one embodiment, the universal reporter peptide U is customized for aspecific mass spectrometer and/or specific use for identification,characterization, and quantitation of disease biomarkers (proteomics,metabolomics, pharmacoproteomics) discovery, confirmation, validation,and early clinical diagnosis and disease progression monitoring. Forexample, a 4-10 amino acid reporter peptide will fragment into fewerproduct ions in a mass spectrometer, yielding greater fragment ionintensities overall than an 11-25 amino acid reporter peptide. The moreintense product ions would give better sensitivity for a triplequadrupole mass spectrometer. Alternatively, a larger 11-25 amino acidpeptide could ionize at a higher charge state and be seen and measuredmore readily with a high mass accuracy mass spectrometer. Proteomics hasadvanced from identification (qualitative proteomics) to quantitation byincorporating an internal standard in the assay. An internal standard isrequired because the resulting peak height or peak surface in massspectroscopy results from a complex function of parameters (e.g.,peptide quantity, peptide ionization, peptide fragmentation, ionsuppression, etc.). There is no algorithm able to evaluate the responsefactor, which is the correlation between the peak surface and quantityof peptide, and therefore to measure the quantity of a peptide from thesurface of the mass spectroscopy peak. When a known quantity of theinternal standard is added to the peptide to be analyzed, the quantityof the peptide is determined by comparing its peak surface with theinternal standard peak surface.

One embodiment is a described peptide modified with a tag. Such a tag,used to modify the peptide, differs from the heavy isotope label that isrequired or is optional to modify universal reporter U and reporter R,respectively. The tag, however, may be a heavy isotope, as subsequentlydescribed in the first example.

One example of such a tag is a heavy isotope. One example of such a tagis an isotopic tag. Such tags include forms of the same chemicalstructure with each tag having an incrementally heavier mass by 5 Da.Upon peptide fragmentation, the tag breaks to release a differentspecific reporter peptide that has a different weight.

One example of such a tag is a different isotope of the same peptide.This example uses as a tag an element that naturally has multipleisotopes, and where the isotopes have a different mass. For example,chlorine may be used because chlorine has two natural isotopes thatdiffer by 2 Da.

One example of such a tag is a mass defect tag. This example uses as atag an element that has a known mass defect such as bromine or fluorine.Use of a mass defect tag shifts the reporter peptide to a region of themass chromatogram in which most isotopes are not observed, sometimesreferred to as a mass quiet space. This example is useful to enhancesensitivity and specificity of detection in a mass region with manyother background ions.

One example of such a tag is a retention time tag. Use of a retentiontime tag shifts the reporter peptide to a region of the masschromatogram in which most peptides are not observed, sometimes referredto as a chromatographic quiet space. This example is useful to enhancegradient efficiency and utility for enhanced separation. As one example,a sample with early elution from a chromatography column uponapplication of a solvent or solvent gradient consumes less time andresources, which enhances efficiency. In use, this embodiment permitsone to determine both the custom peptide and the universal reporterpeptide U by using focused chromatographic conditions in short liquidchromatography analyses, i.e., runtimes less than one minute. As oneexample, a sample with late elution from a chromatography column uponapplication of a solvent or solvent gradient would be expected to havereduced cross contamination because there would be fewer elutingpeptides in this region of the gradient. In all cases, instrument dutycycle is not wasted, and sensitivity and quantitative accuracy are notaffected during critical gradient times. Use of such a focused systemrequires verification with a universal reporter peptide U that isdemonstrated to efficiently and predictably elute under theseconditions.

As one example of customization, the universal reporter peptide U iscustomized for use in stable isotope labeling with amino acids in cellculture (SILAC). Stable isotope-labeled amino acids are fed to livecells and the labeled amino acids are incorporated into polypeptides.The universal peptide is designed to quantify peptides from structuralproteins, chaperones, or housekeeping enzymes to quantify and normalizeprotein quantities between samples. SILAC and its variations, known toone skilled in the art, uses mass spectrometry to quantitate and compareproteins among samples, and sample normalization and measurement ofbiological variation with structural proteins, chaperones, orhousekeeping enzymes allows large numbers of samples to be processed andcompared. In one embodiment, the universal reporter peptide U iscustomized for use with isobaric labeling using either tandem mass tags(TMT) or isobaric tags for relative and absolute quantitation (iTRAQ) bylabeling a set of universal reports for quantitation of peptides fromcommonly observed proteins in cell and tissue lysates, serum and plasma,and formalin-fixed paraffin embedded tissue slices. TMT and iTRAQ havethe general structure M-F-N-R where M=mass reporter region, F=cleavablelinker region, N=mass normalization region, and R=protein reactivegroup. Isotopes substituted at various positions in M and N cause eachtag to have a different molecular mass in the M region with acorresponding mass change in the N region, so that the set of tags havethe same overall molecular weight. Only when the TMT undergo a second orthird fragmentation (such as in tandem mass spectrometry MS/MS, orsequential mass spectrometry MS/MS/MS, MS³) are they distinguishable,with backbone fragmentation yielding sequence and tag fragmentationyielding mass reporter ions needed to quantitate the peptides. iTRAQ andTMT covalently label amine groups in protein digests and a cysteinereactive TMT labels thiols of cysteines, resulting in individual digestswith unique mass tags. The labeled digests are then pooled andfragmented into peptide backbone and reporter ions. The peptide backboneions are used to identify the protein from which they came. The reporterions are used to quantify this protein in each of the combined samples.SILAC, TMT, and iTRAQ mass spectroscopy methods used in biomarkerdiscovery to generate candidate markers are used on instrumentation thatinclude LTQ Velos (Thermo Scientific) and LTQ Orbitrap Velos (ThermoScientific) hybrid mass spectrometer. The candidate markers are thenfurther evaluated and applied in target analysis using selected reactionmonitoring (SRM) to target quantitation of peptide markers in manysamples. For confirmation and validation, the universal reporter peptideU is customized for use, as explained below, with the markers that werepreviously identified, for absolute quantitation with syntheticstable-isotope-labeled peptide standards (HeavyPeptide AQUA and itsvariations, Thermo Scientific) using existing discovery data to automatethe preliminary selection for targeted analysis (Pinpoint software,Thermo Scientific; TSQ Vantage triple stage quadrupole mass spectrometer(Thermo Scientific)). The data are entered into a integrated datamanagement system for clinical applications.

One embodiment is a universal reporter peptide U synthesized to provideit with similar (e.g., ±10% to 20%) properties (e.g., retention time,ionization, optimal fragmentation energy, limit of detection, digestionefficiency, etc.) to a custom peptide. In a method using thisembodiment, the universal reporter peptide U is used to assess digestionefficiency. In use, this embodiment permits one to assess theproteotypic peptide and both the undigested and digested custom peptideand the universal reporter peptide U. The efficiency of digestion of thecustom and universal peptide to the individual peptides is then used tocorrect the level of proteotypic peptide quantified, allowing moreaccurate absolute quantitation of the protein of interest and moreaccurate quantification between samples by correcting for digestefficiency between samples.

One embodiment is a set of universal peptides U. This set of universalpeptides U co-elutes in a predictable manner. The peptides in the setmay or may not share a common sequence. The peptides in the set havestable isotopes incorporated at unique positions to enable specificquantitation of each.

One embodiment is universal reporter U that is not limited to a peptideor that does not include a peptide component at all. This embodimentuses a universal polymer U^(polymer). One example of such anon-peptide-limiting universal reporter U is sequence of natural andnon-natural amino acids. One example of such a non-peptide-limitinguniversal reporter U is a deoxyribonucleic acid (DNA) sequence. Oneexample of such a non-peptide-limiting universal reporter U is a lockednucleic acid (LNA) sequence. One example of such a non-peptide-limitinguniversal reporter U is a peptide nucleic acid (PNA). One example ofsuch a non-peptide-limiting universal reporter U is a threose nucleicacid (TNA). As known to one skilled in the art, a PNA is an artificiallysynthesized polymer. Its structure is similar to the structure ofdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA) but, unlike DNAhaving a deoxyribose triphosphate structure from which bases areattached, or RNA having a ribose triphosphate structure from which basesare attached, PNA has a repeating N-(2-aminoethyl) structure linked bypeptide bonds from which bases are attached by methylene carbonyl bonds.As known to one skilled in the art, a TNA has a repeating threosestructure linked by phosphodiester bonds. As known to one skilled in theart, a LNA is a modified ribonucleic acid (RNA) in which ribose containsan additional bond between its 2′ oxygen and its 4′ carbon, enhancingbase stacking and affecting nucleic acid hybridization by increasingmelting temperature T_(m)).

This embodiment adds to a sample an isotopically labeled proteotypicanalyte coupled to a reporter analyte R through a cleavable site.Cleavage or digestion at the cleavable site decouples the reporteranalyte R from the proteotypic analyte resulting in equimolarconcentrations of each of the proteotypic analyte and the reporteranalyte R in the sample. Mass spectroscopy analysis is performed withoutamino acid analysis to determine a concentration of the reporter analyteR using a quantity of a universal polymer U^(polymer) added to thesample. The universal polymer U^(polymer) has the same amino acid ormonomer sequence, but a different atomic mass, as reporter analyte R dueto the presence of a stable isotope label in universal polymerU^(polymer).

One embodiment is a universal peptide compound, composition,formulation, and/or kit.

In one embodiment, a kit contains a composition of concatenated peptidesand a universal reporter sequence LVALVR (SEQ ID NO. 48) having a purityof about 80%, that is, a relatively low purity. While the analyticalHPLC is relatively low, the fact that the universal reporter sequence ispositioned in the N-terminal region, as shown in FIG. 15, is favorableto a stoichiometry closer to 1:1, which provided a precise method. Thiswas due to synthesis from C— to N— and a capping agent used duringsynthesis. In this embodiment, the kit contained the followingcomponents: 5 aliquots of 0.5 nmol HeavyPeptide IGNIS CCC™ (ThermoFisher Scientific) (polypeptide of N-terminal reporter peptide R andC-terminal heavy isotope labeled proteotypic peptide (A*); 5 aliquots ofdilution curve isotopologues mixture, composition shown in FIG. 19; 5aliquots of universal reporter R bracketing mixture, composition shownin FIG. 20. Mixtures, as known to the skilled person, are the product ofa mechanical blending or mixing of chemical substances (e.g., elements,compounds) without chemical bonding or other chemical change, so thateach component retains its own chemical properties and composition. Asshown in FIG. 15, HeavyPeptide IGNIS™ AAA analytical HPLC purity >80%with an N-terminal reporter R position was more favorable to a 1:1stoichiometry between the reporter R and the proteotypic sequence. Itsstandard length was ≦26 amino acids, including the 6 amino acids fromreporter R. Optionally, additional light amino acids and modifications(S-carboxymethylation of cysteine (CAM), phosphorylation of serine,threonine, and/or tyrosine, etc.) can be added, as known to one skilledin the art.

In one embodiment, the heavy proteotypic peptide—reporter peptide R canbe formulated dry. In one embodiment, the heavy proteotypicpeptide—reporter peptide R can be formulated in solution. The heavyproteotypic peptide—reporter peptide R is stabilized and solubilizationis facilitated by formulating it in a matrix with a non-reducing sugar(e.g., monosaccharides, oligosaccharides, polysaccharides andderivatives, sorbitol, mannitol, trehalose, etc.), or any matrix thatfacilitates reconstitution of the peptide while stabilizing the peptideat ambient temperature (e.g., about 19° C. to about 22° C.) usingmethods known to one skilled in the art. In this form, it is stable atatomole or femtomole quantities. In one embodiment, the heavyproteotypic peptide—reporter peptide R is formulated as a tablet thatcould be transported and stored at ambient temperatures and would beeasily transferred to vials with the need for liquid measurement. Thisformat eliminates concerns about peptide binding nonspecifically to atube wall or solvent evaporation resulting in changes in peptideconcentration. This embodiment reduces the number of manipulationsrequired and, hence, decreases error. This embodiment facilitatesautomation.

In one embodiment, the heavy proteotypic peptide—reporter peptide R isused in food control applications. Universal peptides are used to detectproteins from food allergans, such as almond, egg, gliadin/gluten,hazelnut, lupine, casein, β-lactoglobulin (BLG), total milk, mustard,peanut, sesame, shellfish, soy, and walnut residues, and/or contaminantssuch as Salmonella, E. coli, viruses, protozoan parasites, prions, andother zoonotic diseases, mycotoxins, and aflatoxins. Universal peptidesare used in MS-based tests to detect the target allergen or toxin iningredients, liquids, clean-in-place rinses, finished foods, and/or onenvironmental surfaces.

In one embodiment, the heavy proteotypic peptide—reporter peptide R isused in biobanking. Investigators realize that proper biosamplecollection, processing, storage, and tracking is critical for biomarkerrelated studies. Investigators are concerned that information stored inbiobanks will be inaccurate and incomplete, and thus of questionableusefulness to research, leading to spurious links between proteins,genes, and metabolite biomarkers. The addition of a quality control stepwill quality the condition of any sample used prior to studies on thatsample. Quality control is based on incorporating precise and knownquantities of isotopic peptide standards to a biological sample prior tofinal storage. These peptides have different proteolytic degradationkinetics, are labeled with stable heavy isotopes, and are quantifiablewith existing MS technology. Upon request for a biobanked sample, thequantity of each peptide standard is measured by MS and used to evaluatethe sample quality. Formulation of the heavy peptide-reporter peptide Ris stabilized. Solubilization is facilitated by formulating it with anon-reducing sugar (e.g., sorbitol, mannitol, etc.) using methods knownto one skilled in the art. In this form, the quality control sample isstable at attomole or femtomole quantities.

As one example, when the protein or polypeptide to be quantified isrepresented as A, the internal standard is represented as A*, with *denoting the labeled form; when the protein or polypeptide to bequantified is represented as B, the internal standard is represented asB*, etc. In one embodiment, the internal standard can be anon-proteotypic peptide, e.g., a β-amyloid peptide can be used as aninternal standard to quantify β-amyloid peptide in a biological sample.For example, the following β-amyloid peptides and peptide fragments maybe used; the bolded amino acids indicate mutations in the β-amyloidpeptide sequence:

(1-40) peptide: SEQ ID NO. 20Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val (1-42) peptide: SEQ ID NO. 21Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala (1-43) peptide: SEQ ID NO. 22Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala-Thr (1-46) peptide:SEQ ID NO. 23Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala-Thr-Val-Ile-Val(1-40 F4W) peptide: SEQ ID NO. 24Asp-Ala-Glu-Trp-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val (1-40 Y10W) peptide: SEQ ID NO. 25Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Trp-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val (1-40 D23N) peptide: SEQ ID NO. 26Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asn-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val (1-42 M35V) peptide: SEQ ID NO. 27Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Val-Val-Gly-Gly-Val-Val-Ile-Ala (1-42 R5G) peptide:SEQ ID NO. 28Asp-Ala-Glu-Phe-Gly-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala (1-42 Y10A) peptide:SEQ ID NO. 29Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Ala-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala (1-42 F4W) peptide:SEQ ID NO. 30Asp-Ala-Glu-Trp-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala (1-42 H6A) peptide:SEQ ID NO. 31Asp-Ala-Glu-Phe-Arg-Ala-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala (1-42 H13A) peptide:SEQ ID NO. 32Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-Ala-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala (1-42 H14A) peptide:SEQ ID NO. 33Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-Ala-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala (1-11) peptide fragment:SEQ ID NO. 34 Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu(1-16) peptide fragment: SEQ ID NO. 35Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys(1-28) peptide fragment: SEQ ID NO. 36Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys (1-38) peptide fragment:SEQ ID NO. 37Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly (11-22) peptide fragment: SEQ ID NO. 38Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu(11-42) peptide fragment: SEQ ID NO. 39Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile- (11-40) peptide fragment: SEQ ID NO. 40Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val(12-28) peptide fragment: SEQ ID NO. 41Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn- Lys(17-40) peptide fragment: SEQ ID NO. 42Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val (17-42) peptide fragment: SEQ ID NO. 43Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala (22-35) peptide fragment:SEQ ID NO. 44 Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met(25-35) peptide fragment: SEQ ID NO. 45Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met (1-40) peptide.

EXAMPLE 1

Stable isotope labeled peptides containing a universal reporter peptideU and several peptides concatenated was applied to detect and quantifyprotein biomarkers in clinical samples, with a focus on markers of lungcancer.

To assess the recovery of the sample preparation method, heavyisotopically labeled synthetic polypeptide standards (comprising up tothree proteotypic peptides and a universal reporter U) of human plasmaproteins (LDH, NSE, and Myo) were spiked in samples before and afterproteolysis. HPLC-MS analyses were performed on a triple quadrupoleinstrument (TSQ Vantage, ThermoFisher Scientific) in SRM mode. A set ofconcatenated reference peptides was synthesized based on a list ofcandidates previously identified. Synthetic polypeptides were obtainedfrom ThermoFisher Scientific (Ulm Germany).

The reporter peptide R was designed with a tryptic cleavage site at theC-terminus. The calibration curves for the universal reporter peptides Uwere established using dilution series. The relative response factor ofeach peptide compared to the reporter was readily determined aftertrypsin treatment exploiting the 1:1 stoichiometry.

A panel of proteins indicative of lung cancer was selected todemonstrate proof-of-principle. For precise quantification of specificproteins, three synthetic concatenated proteotypic polypeptides weregenerated and analyzed. Plasma samples from lung cancer patients andcontrols were analyzed.

The concatenated synthetic polypeptides containing a universal reporterenabled precise determination of the quantities of targeted proteinspresent in the sample using concomitantly multiple reference peptides.This quantification approach was readily implemented in a large scaletargeted proteomics workflow.

EXAMPLE 2

Stable isotope labeled peptides containing a universal reporter peptideU and several peptides concatenated were used to detect and quantifyprotein biomarkers in clinical samples, with a focus on markers ofbladder cancer.

Exogenous proteins from yeast (Saccharomyces cerevisiae) (ADH, enolase,and carboxypeptidase) and human (LDH, NSE, Myo) were added as internalstandards in urine samples, which may be prepared as shown in FIG. 23.The isotopically labeled synthetic polypeptide standards, which wereproteotypic peptides of the protein of interest and a universal reporterpeptide U, were spiked before proteolysis. Urine samples were preparedby protein precipitation, reduction/alkylation, trypsin proteolysis, anddesalting using C18 cartridges. A second set of isotopically labeledsynthetic peptides was added after proteolysis.

LC-MS/MS analyses were performed on RP-HPLC (Dionex) coupled with atriple quadrupole instrument (TSQ Vantage, ThermoFisher Scientific)operated in SRM mode. Several transitions were monitored for eachtargeted peptide. Isotopically labeled synthetic polypeptides wereobtained from ThermoFisher Scientific (Ulm Germany).

To establish the methodology, stable isotope-labeled dipeptidescontaining a universal reporter U were synthesized. The reporter peptideR was designed with a tryptic cleavage or digestion site at theC-terminus. The calibration curves for the universal reporter peptides Uwere established using dilution series. In parallel, the relativeresponse factor of each peptide compared to the reporter was determinedafter trypsin treatment exploiting the 1:1 stoichiometry. LC-MS analyseswere performed in SRM mode.

To evaluate the methodology, precise quantities of referencepolypeptides were spiked into the urine samples, digested with trypsin,and analyzed by LC-SRM to quantify the targeted human proteins.Preliminary results included analysis of insulin-like growth factorbinding protein 7 in urine samples using two individual synthetic stableisotope-labeled peptides tagged with a universal reporter U:reporter-HEVTGWVLVSPLSK (SEQ ID NO. 46) and reporter-ITWDALHEIPVK (SEQID NO. 47). Dilution curves were generated in pooled urine samples toprecisely determine the quantity of corresponding protein: the actualconcentrations obtained were 1.49 ng/ml and 1.69 ng/ml, for the twopeptides respectively.

EXAMPLE 3

To further evidence the method's utility, large synthetic polypeptideswere produced, resulting from concatenation of multiple peptidesrepresenting each of the proteins of interest. Three proteotypicpeptides per protein with adequate mass spectrometric properties(precursor m/z, ionization efficiency, retention time, MS/MS spectra)were selected to construct the concatenated standards. These referencepolypeptides, all containing a universal reporter, allowed measurementof the precise quantity of multiple reference peptides in one LC-MS run.

EXAMPLE 4

The inventive method decreased mass spectrometry usage to generate acalibration curve, resulting in savings in both instrument usage,operator time, and processing efficiency. In preparing a calibrationcurve, different peptide concentrations are injected in the LC-MSsystem. A typical calibration curve requires six peptide injections atdifferent peptide concentrations. Each injection is performed intriplicate (three replicates). The total LC-MS analyses to generate acalibration curve is thus at least 18 analyses.

The peptide LVALV*R*SEQ ID NO. 48 was injected (* indicates a heavyisotope labeled amino acid). The following peptides isomers of SEQ IDNO. 48 were synthesized: LVALVR*, LV*ALV*R*, LVA*L*V*R*, LV*A*L*V*R*,L*V*A*L*V*R* and LVA*LVR (other combination are possible) and providedin equimolar concentration. Two mixtures of these synthesized peptideswere prepared. Each mixture had a different concentration of heavyisotope: one mixture had a three fold or three times dilution of eachisomer; the other mixture had a five fold or five times dilution betweenof each isomer. The final mixtures containing the isomers at differentconcentrations were injected into the LC-MS system. A single injection,with three replicates, was sufficient to generate a complete calibrationcurve. This method thus further decreased the time needed to generatethe calibration curve, and also improved the coefficients of variationamong the three replicates.

EXAMPLE 5 Isotopically-Labeled Peptides as Internal Standards

Table 1 lists targeted human urine and yeast proteins, and threeselected proteotypic peptides to design HeavyPeptide IGNIS™. As shown inTable 1, ten HeavyPeptide IGNIS™ were designed that corresponded to 30proteotypic peptides of nine proteins (seven human proteins, three yeastproteins). Stable isotope-labeled amino acids of each HeavyPeptideIGNIS™ (purity >95%, lyophilized in sorbitol) were selected to have amass change sufficient for MS analysis, with the endogenous peptides andthe corresponding individual synthetic stable isotope labeled peptideswith C-terminal ¹⁵N- and ¹³C-labeled arginine or lysine residue (purity,lyophilized, >99 atom % isotopic enrichment); Table 2 lists the fullsequence of the HeavyPeptide IGNIS™ identifying the stable isotope aminoacids.

The proteotypic peptides were selected from proteomics shotgunexperiments. The reported number of observations was used as a surrogateindicator for the abundance of proteins in a specific proteome. Theuniqueness of the peptide reporter was verified by blasting the aminoacid sequence, LVALVR, against the UniProt database; this sequence isnot associated with a protein.

Calibration Curve of the Peptide Reporter

The calibration curve was performed by mixing the universal reporterpeptide U solution (purity >97%) with various isotope label (R for heavyarginine (¹³C₆, ¹⁵N₄); A for heavy alanine (¹³C₃, ¹⁵N); L for heavyleucine (¹³C₆, ¹⁵N); V for heavy valine (¹³C₄, ¹⁵N)). Five μL of LVALVR(0.5 fmol/μL), 15 μL LVALVR (0.5 fmol/μL), 4.5 μL LVALVR (5 fmol/μL),13.5 μL LVALVR (5 fmol/μL), 40.5 μL LVALVR (5 fmol/μL), 12.2 μL LVALVR(50 fmol/μL), 36.5 μL LVALVR (50 fmol/μL), 109.4 μL LVALVR (50 fmol/μL),and 13.6 μL of 0.1% (v/v) formic acid (in water) were mixed to obtain afinal volume of 250 μL. Concentrations of these peptides in solution are10.0 atmol/μL, 30.0 atmol/μL, 90.0 atmol/μL, 270.0 atmol/μL, 810.0atmol/μL, 2.4 fmol/μL, 7.3 fmol/μL and 21.9 fmol/μL, respectively.Analysis of the calibration curve was performed in triplicate by oneLC-SRM run on the TSQ-platform. The two most intense transitions derivedfrom the SRM assay were monitored for the reporter peptide.

The results of the “dilution series” of the universal reporter peptideU=LVALVR are shown in FIG. 9. Various isotopic labels, ranging from 0.01fmol (injected)-21.9 fmol (injected) are shown in linear (FIG. 9A) andlogarithmic scales (FIG. 9B). The dilution series was measured in onesingle LC-MS run. Peak area ratios were determined from the mean of twoSRM transitions. Each data point corresponds to the dilution series thatwas measured in triplicate. A linear standard curve was observed(r²>0.999) between 10.0 atmol/μL and 21.9 fmol/μL.

Proteolysis of HeavyPeptide IGNIS™

Each HeavyPeptide IGNIS™ was solubilized with acetonitrile (ACN)/water(15/85) (vol/vol) to obtain a final protein concentration of 5 pmol/μL,and then sonicated for 20 minutes. HeavyPeptide IGNIS™ were individuallydigested by trypsin 1:20 (w/w) (Promega, Madison Wis.) for 3.5 hr at 38°C. under agitation (1400 rpm). The kinetic digestion was monitored byreaction mixture extraction every 15 minutes. To stop the digestion, allsamples were diluted in 0.1% v/v formic acid to obtain a final peptideconcentration of 50 fmol/μL for analysis on LC-MS (in SRM mode).

For quantitative measurements on TSQ-platform, all HeavyPeptide IGNIS™digestion kinetic points were stoichiometrically supplemented with thecorresponding individual synthetic stable isotope-labeled peptides withC-terminal ¹⁵N/¹³C-labeled arginine or lysine residue (AquaUltimate) andthe universal reporter peptide U=LVALVR. The two most intensetransitions observed from the SRM assay were monitored for all peptides.FIG. 10 shows proteolysis efficiency of the HeavyPeptide IGNIS™ UROM.Proteolytic kinetics of HeavyPeptide IGNIS™ UROM was established bynonlinear regression of each heavy proteotypic peptide (UROM A, UROM B,and UROM C) from digested HeavyPeptide IGNIS™ UROM/universal reporterpeptide U area ratio versus time of the proteolysis. The last points ofkinetic were used to define the precise quantity of each HeavyPeptideIGNIS™.

Urine Collection and Sample Treatment

Spot midstream urine samples were collected from ten non-smoking healthyvolunteers, five females and five males, age range 30-40 years. Therewas no history of renal dysfunction in any of the subjects or drugadministration during the sample collection. Urine was centrifuged at 1000 g relative centrifuge force (rcf) per 20 minutes at room temperature(about 19° C.-22° C.). The supernatants 1 000 g were pooled together,portioned into aliquots in 50 Falcon™ tubes and stored at −80° C.

The quantity of urinary protein was estimated by a pyrogallol assay.Samples corresponding to 250 μg of urinary protein were precipitatedwith 100% stock solutions of acetonitrile (for HPLC) at a ratio 1:5(v/v). Samples were incubated at room temperature overnight. Afterprecipitation, urine samples were centrifuged at 14,000 g for 30 minutesat 4° C. The pellet was washed once with the acetonitrile, air-dried.and resuspended with 250 μL 8 M urea and 0.1 M ammonium bicarbonate. Thesamples were reduced with 20 mM dithiothreitol in 50 mM ammoniumbicarbonate at 37° C., centrifuged at 800 rpm for 30 minutes, thenalkylated with 80 mM iodoacetamide in 50 mM ammonium bicarbonate at 37°C. and centrifuged at 800 rpm for 30 min. Volume samples were adjustedat 2 M urea with 100 mM BA. Samples were then digested with trypsin(Promega, Madison Wis.) using a ratio of 1:20 (w/w) at 37° C. overnight.Digestion was halted by adding formic acid to obtain a pH 2-3. Sep-PakC18 reverse phase cartridges, 100 mg (Waters, Milford Mass.) were usedto clean and desalt the samples after protein digestion. The peptideswere eluted using 1 mL of 50% acetonitrile and 0.1% formic acid, dried,and stored at −20° C. until LC-MS analysis.

Calibration Curve of HeavyPeptide IGNIS™ and AquaUltimate in UrineSamples

Dilution curves of the heavy proteotypic peptides from digestedHeavyPeptide IGNIS™ were performed in a mixture of digested pooled urinesample (1 ug/mL urine proteins), containing three digested exogenousyeast proteins (carboxypeptidase Y, enolase 1, and alcoholdehydrogenase 1) at 100 ng/mL individually. Each dilution seriescorresponded to three data points spanning a concentration ranging from0.002 fmol/uL to 40 fmol/uL. Protein levels of spiked digested yeastproteins and human urine proteins were determined by iSRM using theheavy proteotypic peptides from digested HeavyPeptide IGNIS™. FIG. 11shows a dilution curve of the heavy proteotypic peptides from thedigested HeavyPeptide IGNIS™ KNG1 from 0.002 fmol/μL to 40 fmol/μL on alogarithmic scale. Peak area ratios were determined from the mean to twoSRM transitions. Each data point corresponded to the dilution seriesthat was measured in triplicate. FIG. 11 and Table 3 show data for theHeavyPeptide IGNIS™ Kininogen 1 (KNG 1).

LC-MS Conditions

Urinary and yeast tryptic peptides were analyzed on a TSQ Vantage TripleQuadrupole Mass Spectrometers (ThermoFisher, San Jose Calif.).Instruments were equipped with a nanoelectrospray ion source.Chromatographic separations of peptides were performed on an Ultimate3000 (Dionex, Netherlands) high performance liquid chromatographeroperated in the nano-flow mode. Samples were loaded on a Trap column(Acclaim PepMap C18, 3 μm, 100 Å, 0.075×20 mm, Dionex) and separated onan analytical column (Acclaim PepMap® RSLC C18, 2 μm, 100 Å, 0.075×150mm, Dionex) coupled with a PicoTip™ electrospray emitter (30 μm) (NewObjective, Woburn Mass.) maintained at 1.2 kV. The column temperaturewas fixed at 35° C. Peptides were separated with a linear gradient ofacetonitrile/water, containing 0.1% formic acid, at a flow rate of 300mL/min. A gradient from 2% to 35% acetonitrile in 33 minutes was used.One μL of each sample was injected. The mass spectrometer was operatedwith intelligent-selected reaction monitoring (i-SRM). For acquisitions,the mass selection window of Q1 and Q3 were 0.7 unit mass resolution. Anacquisition time window of 2 min was set around the elution time of thepeptide. Argon was used as the collision gas at a nominal pressure of1.5 mTorr. Collision energies for each transition were calculatedaccording to the following equations: CE=0.033*(m/z)+1.8 andCE=0.038*(m/z)+2.3 (CE, collision energy and m/z, mass to charge ratio)for doubly and triply charged precursor ions, respectively. For thei-SRM acquisition, two primary and six secondary fragment ions wereselected per peptide, based on the full MS/MS spectrum stored in adatabase. The scan time for the triggered SRM measurements was 200 ms.Dynamic exclusion for the triggering of the data-dependent SRM wasenabled with a threshold intensity of 100 counts, with a repeat count ofthree cycles and a dynamic exclusion of 20 seconds. The i-SRM data wereprocessed using Pinpoint software (Thermo Fisher, San Jose Calif.).

EXAMPLE 6

Table 4 lists identifications and characterizations for selected humanand yeast HeavyPeptide IGNIS™ peptides used as disclosed. The peptideswere in a lyophilized formulation, purity >95%. The universal reporterpeptide U was LVALVR, with a mass difference (delta mass) versus thenatural sequence=16. Table 5 lists commercially available peptideshaving the same sequence but having a different mass due to a differencein heavy labeled amino acid, that may be used as additional peptides inthe disclosed method.

EXAMPLE 7

Sensitivity limits included both limits of detection (LOD), and limitsof quantitation (LOQ). Variability in LOD and LOQ is due to multiplefactors, including but not limited to different liquid chromatographyequipment and conditions, material, etc.). The common source isnonspecific binding of the peptide to the tubing, column, etc. The LODand LOQ are traditionally determined by injecting more and more dilutedpeptides into the system. The lower the concentration, the higher thechance to lose part or all the peptide.

The dilution series of Example 5 was used to assess sensitivity limitsof MS equipment. Specifically, the following sequences were included inthe mixture: LVALVR, LVALVR, LVALVR, LVALVR, LVALVR, and LVALV. Themixture contained a blend of different isomers, termed isotopologues,each having a slight mass shift, and each present in a differentconcentration, such that the entire calibration curve was generated inone single LC analysis or run.

In one evaluation of quantification performance, the following resultswere obtained from a peptide composition that was a blend or mixture ofeight peptides and each peptide in the composition had the identicalamino acid sequence but different amino acids in the peptide werelabeled or modified with a stable heavy isotope. Each isomer in theblend had a mass shift, and each isomer in the blend was present in adifferent concentration. The dilution spanned a 3.3 logarithmic range,specifically, from 10 amol to 20 fmol. For data acquisition theresolution was 70,000; the maximum injection time was 250 ms, and theautomatic gain control (AGC) target values were 1 E5, 5E5, 3E6. Theresults are shown in FIG. 12 and in the following table.

m/z [M + 2H]²⁺ Peptide Quantity 335.73 LVALVR   10 amol 337.74 LVALVR  30 amol 340.74 LVALVR   90 amol 343.74 LVALVR  270 amol 347.25 LVALVR 810 amol 349.26 LVALVR  2.4 fmol 352.26 LVALVR  7.3 fmol 355.77 LVALVR21.9 fmol

Data supporting use of a dilution curve mixture or blend for singleinjection dilution curve generation are shown in FIG. 16. Thedistribution of medium and low abundant protein spread from 1 pmol per100 μl sample to <1 amol per 100 μl sample. To cover 80% of themedium-to-low abundance proteins, the dilution curve mixture (DCM)should cover up to 0.1 pmol per 100 μl of the final sample. The DCM islyophilized so it can be reconstituted in any buffer, including complexmatrices such as plasma, urine, other body fluid, or cell extract.Reconstituting the DCM in 900 μl, compatible with the volume of astandard vial, provides 3 amol per 1 μl for the lowest concentratedisomer. FIG. 17, providing data from a dilution curve mixture, gives theconcentration per μl for the lowest concentrated and highestconcentrated isomer mixtures or blends. Each peptide had >97% purity. Toprepare the dilution curve mixture, 45 μl of the isomer mixture or blendin 50 mM sorbitol was lyophilized in standard glass vials. Informationon the isomers, including sequences and concentrations, used to generatethe dilution curve mixture is provided in FIG. 18. In one embodiment,the isotopologue mixture or blend contains the isomers listed in FIG. 19and the sequences listed in FIG. 20 as internal standards bracketingcontrols.

Various secondary ion mass (SIM) methods were evaluated as furtherdescribed with results shown in FIGS. 13A, 13B, and 13C. Method A usedone isolation window (30 m/z), a cycle time of about 0.3 s, and onedetection scan; the results are shown in FIG. 13A. Method B used eightisolation windows (2 m/z), a cycle time of about 3.2 s, and eightdetection scans; the results are shown in FIG. 13B. Method C used eightisolation windows (2 m/z), a cycle time of about 1.7 s, and onedetection scan; the results are shown in FIG. 13C.

The results showed that the method resulted in enhanced sensitivity. TheLOQ were in the amol range. The intra-scan linearity exceeded a 3.5logarithmic range, from 10 amol to 25 fmol.

This method avoided the loss of peptide problem (e.g., nonspecificbinding to the column, etc.) because all the isomer mixtures, having thesame peptide sequence, co-eluted. The concentration of the most abundantisomer was higher than the concentration of the most diluted isomer,decreasing the loss of the less abundant isomer by non-specific binding.The concentration of isomer at the highest concentration of peptide was1000, 3000, or 10000 times higher than the concentration of isomer atthe lowest concentration of peptide. The risk of losing the isomer withthe lowest concentration was very low. This mixture was ideal to compareLOD and LOQ from instrument to instrument, brand to brand, and/or day today on same instrument, and resulted in more robust quantitative MS.

EXAMPLE 8

Production of a relatively long universal reporter (HeavyPeptide IGNIS™)(≧40 amino acids), was facilitated using linkers and chemical ligation.Because it is challenging to synthesize and purify such relatively longpeptides, it is often easier to synthesize and purify shorter peptidesand assemble these fragments into the final polypeptides. The assemblyis done through chemical ligation or using “Click Chemistry”, defined asusing reactions that are high yielding, wide in scope, creating onlybyproducts that can be removed without chromatography, arestereospecific, are simple to perform, and can be conducted in easilyremovable or benign solvents. In both cases a sequence “linker” is addedbetween one or more of the proteotypic peptide as the necessary moleculefor the chemical ligation (Cys) or the “click Chemistry” molecule. Thelinker may be an amino acid sequence, poly(ethylene)glycol (PEG), or anyother molecule that can be synthesized as a polymer, as is known to oneskilled in the art. When the linker is an amino acid sequence, in oneembodiment, the natural flanking regions on the endogenous peptideanalyzed are used as a surrogate of protease digestion efficiency, asshown schematically in FIG. 14.

EXAMPLE 9

Stable heavy isotope labeled proteins containing a reporter weresynthesized. Two different patterns of isotopically labeled universalreporters U were used. Linear standard curves were obtained (r²=0.999)by monitoring the three most intense selected reaction monitoring (SRM)transitions. The relative response factor of each peptide, compared tothe reporter R, was determined after proteolysis (1:1 stoichiometry).The results are shown in FIG. 22 with two different reporters R and twoheavy proteotypic peptides for one protein using the same reporter R.

EXAMPLE 10

Data for a dilution series to generate a calibration curve using auniversal peptide U are shown in FIG. 26A and FIG. 26B, respectively.The universal peptide U was LVALVR SEQ ID NO. 48; various heavy isotopelabels in quantities ranging from 10 amol to 20 fmol were injected. FIG.27 is a graph of these data with linear (FIG. 27A) and logarithmic (FIG.27B) scales. The dilution series was measured in one single LC-MSanalysis or run. Peak area ratios were determined from the mean of threeSRM transitions in triplicate measurements.

EXAMPLE 11

Quality control of the non-digested heavy labeled peptides was performedusing uromodulin (UROM) DWVSWTPA*RDSTIQW*ENGESSQGRSGSV*IDQSRLVALVR* (SEQID NO. 53). Results are shown in FIGS. 28-31. The chromatogram and fullMS spectra are shown in FIG. 28. The results of a typical exampledemonstrating the limits of detection (LOD) and limits of quantitation(LOQ) using the digested peptides indicated, and the correspondingprotein quantities measured in urine by i-SRM experiments, are shown inFIG. 29. Calibration curves for the indicated protein in pooled urinesamples are shown in FIG. 30 in linear scale, and FIG. 31 in logarithmicscale.

Sensitivity limits include both limits of detection (LOD) and limits ofquantitation (LOQ). The inventive method and kit have application foruse with next generation mass spectrometers using specific dataacquisition methods, specific data processing methods, relatedinstrumentation, etc.

Data processing allows peptides and related isoforms to be grouped,using specific m/z values for precursors and product ions todifferentiate signals from each isoform. Such grouping permits relativeand absolute quantitation.

In one embodiment, a data processing system is used to analyze anddetermine relative and/or absolute quantities of targeted andnon-targeted biopolymers in a biological sample. The method is based onidentifying, grouping, and targeting a series of biopolymers related bysequence and/or composition. The targets contain identical sets andorders of amino acid residues that differ by heavy isotopic labeling,side chain modifications, and/or N- or C-terminal modificationsresulting in distinct analytical targets separated by m/z values easilydistinguished by a mass spectrometer. For related targets, the m/zvalues are grouped based on, e.g., injection time, dwell time, automaticgain control (AGC) value, and/or charge density in the curved linear iontrap (C-trap). The data acquisition, processing, and reporting schemescan be performed for precursor and/or product ions (MSn) to determine aquantified response for the endogenous as well as the labeledbiopolymers; the product ions are labeled using conventional termsdefined in classical mass spectrometry (e.g., b-, y-, c-, z-, etc.), asknow to the skilled person, to define the specific position of cleavageand site of charge retention. Data acquisition can include determiningthe targeted biopolymer sequences, differentiating factors, dataacquisition strategies, and data processing approaches that can quantifytargeted biopolymers using relative and/or absolute quantities for onesample or across a number of samples.

FIG. 32 schematically illustrates a computer system 1200 that can beused with the method. The computer system 1200 can be a laptop, desktop,server, handheld device (e.g., personal digital assistant (PDA), smartphone, etc.), programmable consumer or industrial electronic device. Asshown, the computer system 1200 includes a processor 1202, which can beany various available microprocessors, e.g., the processor 1202 can beimplemented as dual microprocessors, multi-core and other multiprocessorarchitectures.

The computer system 1200 includes memory 1204, which can includevolatile memory and/or nonvolatile memory. Nonvolatile memory caninclude read only memory (ROM) for storage of basic routines fortransfer of information, such as during computer boot or start-up.Volatile memory can include random access memory (RAM). The computersystem can include storage media 1206, including but not limited tomagnetic or optical disk drives, flash memory, and memory sticks.

The computer system 1200 incorporates one or more interfaces 1208,including ports (e.g., serial, parallel, PCMCIA, USB, and FireWire) orinterface cards (e.g., sound, video, network, etc.) or the like. Inembodiments, an interface 1208 supports wired or wirelesscommunications. Input is received from any number of input devices(e.g., keyboard, mouse, joystick, microphone, trackball, stylus, touchscreen, scanner, camera, satellite dish, another computer system, etc.).The computer system 1200 outputs data through an output device, such asa display (e.g. CRT, LCD, plasma, etc.), speakers, printer, anothercomputer or any other suitable output device.

FIG. 33 schematically shows an exemplary computing environment for thedisclosed systems and methods. The environment includes one or moreclients 1300, where a client 1300 may be hardware (e.g., personalcomputer, laptop, handheld device, or other computing devices) orsoftware (e.g., processes or threads). The environment also includes oneor more servers 1302, where a server 1302 is software (e.g., thread orprocess) or hardware (e.g., computing devices), that provides a specifickind of service to a client 1300. The environment can support either atwo-tier client server model as well as the multi-tier model (e.g.,client, middle tier server, data server and other models).

The environment also includes a communication framework 1306 thatenables communications between clients and servers. In one embodiment,clients correspond to local area network devices and servers areincorporated in a cloud computing system. A cloud is comprised of acollection of network accessible hardware and/or software resources. Theenvironment can include client data stores 1308 that maintain local dataand server data stores 1310 that store information local to the servers1302.

In one embodiment, the method groups the following two sets of peptidesinto a sample for data analysis: a set of exogenous polypeptides relatedby identical amino acid sequences containing different numbers of heavylabeled isotopes, and a set of endogenous polypeptides related by thesame amino acid sequences as defined by the exogenous group and lackingheavy labeled isotope addition. The method then calculates a quantity ofeach of the exogenous polypeptides and the endogenous polypeptides byprocessing the data using a processor. The processor automaticallygroups peptides containing identical amino acid sequences into groupsdifferentiated by heavy labeled isotope inclusion, side chainmodifications, and/or terminal modifications. For a sample, itdetermines a response for each exogenous polypeptide target, andcorrelates the response for each exogenous peptide as a function of aknown quantity that generates a mathematical response linking a measuredmass spectrometry signal and a quantity applied to the massspectrometer; it determines the response for each endogenous polypeptidetarget, and calculates an estimated endogenous polypeptide target basedon the mathematical expression correlated by the exogenous polypeptideanalog. The steps are repeated for the remaining samples based on thecorrelated mathematical expression relating measured signal and/or AUCto an amount or quantity. Specifically, the measured ion intensity forthe specified peptide amount on column or analyzed is plotted,generating generally linear results and permitting the linear responseof the mass spectrometer for the targeted peptide to be calculated. Themethod then determines the endogenous amount by measuring the ionintensity or AUC, depending upon how the exogenous signal was measured.The amount is then entered into the linear equation as defined by theexogenous peptides to determine the amount on the column.

As disclosed, using the inventive universal reporter peptides, peptidesequences to be analyzed by MS methods are concatenated into apolypeptide analog. These concatenated subunits represent anisotopically labeled endogenous targeted peptide, labeled A* or A,belonging to a targeted protein, and the reporter peptide R that may ormay not be isotopically labeled. Because the peptides are synthesized ata 1:1 ratio, upon enzymatic digestion, the signals can be referenced andabsolute quantitation can be performed. Reporter peptide R has adifferent sequence than the endogenous peptides. Reporter peptide Rcontains the exact same sequence as the universal peptide U, which isdifferentiated from R by changing the number of isotopic labels from R.For example, with reporter peptide R having sequence TASEFDSAIAQDK whereamino acid K is isotopically labeled, universal peptide U sequence couldbe TASEFDSAIAQDK where amino acids F and K are isotopically labeled.

For each of (i) the isotopically labeled endogenous targeted peptide A*or A, (ii) reporter peptide R, and (iii) universal peptide U, there areat least two m/z values that are targeted and that can be acquired usingdifferent mass spectral strategies. Each of the isoform pairs, ormultiples, co-eluted.

The co-eluted isoform pairs, or multiples, were grouped for dataacquisition using, in one embodiment, Q Exactive®, a bench-top highresolution quadrupole FT (Fourier transform)-MS system. Q Exactive® isbased on existing high performance quadrupole and Exactive® bench-topOrbitrap technologies (Thermo Fisher Scientific) for ion detection, withan ion source interface (SRIG) lens, a quadrupole mass filer, and animproved high energy collision dissociation (HCD) cell with the Orbitrapanalyzer; it also contains a higher scanning speed through optimizationof parallel operation, multiplexed detection, and transient processing.Its ion source interface is a hybrid FT-MS fitted with a progressivestacked ring ion guide (SRIG) that provides better signal with lessnoise. Its quadrupole mass filter enables precursor ion selection beforethe curved linear ion trap (C-trap) cell, using four hyperbolic rods,r₀=4 mm. Fragmentation for true MS/MS operation is conducted in HCDcell, followed by C-trap ion injection into the Orbitrap for highresolution accurate mass detection; resolving power of the mass filteris 0.4 Da for ions <m/z 400, allowing highly specific precursorselection. The following table provides isolation widths at variablemass ranges:

 50 < m/z < 400 any isolation widths between 0.4 amu and full scan 400 <m/z < 700 any isolation widths between 0.7 amu and full scan  700 < m/z< 1000 any isolation widths between 1.0 amu and full scan 1000 < m/z <2000 any isolation widths between 2.0 amu and full scan >2000 m/z noprecursor ion isolationThere is multiplexed detection; more than one, and up to 10, isolatedprecursor ions are collected before Orbitrap detection, significantlyenhancing the duty cycle. The multiple secondary ion mass (SIM) modeenhances the duty cycle by a factor of N−1×transient acquisition time(N=number of precursors in one multiplex experiment). There is enhancedFT transient processing using an algorithm that enhances the resolvingpower by a factor of 1.8, yielding higher resolutions at higheracquisition speeds. The following table shows respective resolving powersettings:

Transient Resolving Power actual scan length (ms) at m/z 200 speed (Hz)64 17.500 12 128 35.000 7 256 70.000 3 512 140.000 1.5The transfer optics and HCD collision cell are incorporated into acombination C-Trap and HCD collisions cell, providing an enhanced dutycycle for HCD MS/MS scans and improved low mass ion transmission to theOrbitrap. HCD transmission for ITraq and TMT tags are improved,resulting in improved S/N and quantitation in proteomic assays.

Using Q Exactive®, the quadrupole mass filter passes ions throughtowards the HCD cell and C-trap cell and can be set to filter discretemass ranges. One setting passes a very narrow mass window (1 Da or 2 Da)centered on the individual isoform sequentially, and collecting in theC-trap cell prior to detection. One setting passes one limited massrange (10 Da) centered around the average mass value for the isoformgrouping. One setting passes one larger mass range (>10 Da) toquantitate multiple groups simultaneously. One setting passes oneextremely large mass range (200 Da-1000 Da) simulating a full scan MSanalysis.

The HCD cell can be operated in high or low vacuum (relative) to eithercreate low energy or high energy data collection. The C-trap collects,focuses, and injects into the orbitrap; the orbitrap is used for FTdetection. FT detection simultaneously detects all ions in the orbitrapregardless of m/z value, it thus detects and quantitates the targetedpeptides and their heavy labeled analogs. The quadrupole mass filterenables the Q Exactive® to pass a discrete mass range from the ionsource region to the HCD cell, C-trap cell, and orbitrap massspectrometer. The filtered mass window ranges from 0.4 Da to 4 Da, 10Da, 50 Da, 100 Da, 200 Da, 500 Da, and larger, depending on the desiredfiltering window. By filtering the mass range transmitted from thesource to the detector, the instrument increases detection capabilitiesby reducing background matrix ions from the orbitrap, thus increasingthe charge density attributed to the targeted peptides.

The method of collecting targeted mass windows can be sequential, butdetection and measuring ion abundance are simultaneous. This isdistinguished from ion traps, triple quads, and TOF systems that do notcollect discrete mass ranges prior to detection and do not havesimultaneous detections. The latter instruments collect ions over eitherdiscrete mass ranges or detect over discrete mass ranges, but not both.

The method of ion accumulation is key to normalize the spectrum andrelate ions of different abundance values. Targeted ions, both unlabeledand labeled analogs, are grouped in the instrument method editor priorto detection. This grouping links all aspects of data acquisition suchas detection times, scheduled acquisition, automatic gain control,maximum ion fill times, etc., for improved evaluation. Setting theautomatic gain control better normalizes the resulting data and providea way to assign absolute quantities based on the spiked in levels of thesynthetic peptides.

The method permits ion collection using low and/or high energy tocollect and quantitate precursors ions or product ions. MS-level (lowenergy) is used for precursor detection and/or quantitation. MSn-level(high energy) (MSn denoting MS/MS, MS³, etc.) is applicable to bothendogenous and exogenous targets for quantitation and is used forproduct ion detection and measurement. Reducing the gas pressure in theHCD cell, and changing the voltage, activates ions passed through thequadrupole mass filter. If the mass filter is set to pass 2 Da windowscentered around each independent member of a targeted group, theresulting product ions are independently formed. The user has the optionto measure the tandem MS data independently, or to collect all productions from the group and perform simultaneous detection. The mass filtercan be set to pass wider mass ranges to simultaneously activate all ionsas well as collect and measure ion abundance values.

Applicants incorporate by reference the material contained in theaccompanying computer readable Sequence Listing entitled“078672_(—)4.txt”, which was created on Jul. 6, 2011 and is 32,125 bytesin size.

TABLE 1  Heavy Peptide IGNIS ™ SwissprotSelected proteotypic peptides (PI, PII, PIII) Name Protein Name OrganismID P I P II P III uromodulin human P07911 DWVSVVTPAR DSTIQVVENGESSQGRSGSVIDQSR (SEQ ID NO. 49) (SEQ ID NO. 50) (SEQ ID NO. 51) TRFEserotransferrin human P02787 DGAGDVAFVK SASDLTWDNLK EGYYGYTGAFR(SEQ ID NO. 54) (SEQ ID NO. 55) (SEQ ID NO. 56) LG3BP* galectin-3- humanQ08380 LADGGATNQGR SDLAVPSELALLK ELSEALGQIFDSQR binding (SEQ ID NO. 58)(SEQ ID NO. 59) (SEQ ID NO. 60) protein CD44 CD44 antigen human P16070FAGVFHVEK YGFIEGHVVIPR ALSIGFETCR (SEQ ID NO. 62) (SEQ ID NO. 63)(SEQ ID NO. 64) CATD cathepsin d human P07339 LVDQNIFSFYLSR VSTLPAITLKYSQAVPAVTEGPIPEVLK (SEQ ID NO. 66) (SEQ ID NO. 67) (SEQ ID NO. 68) KNG 1kininogen-1 human P01042 TVGSDTFYSFK YFIDFVAR YNSQNQSNNQFVLYR(SEQ ID NO. 70) (SEQ ID NO. 71) (SEQ ID NO. 72) ANAG alpha-N-acetyl-human P54802 LLLTSAPSLATSPAFR YDLLDLTR SDVFEAWR glucosaminidase(SEQ ID NO. 74) (SEQ ID NO. 75) (SEQ ID NO. 76) ENO1 enolase 1 yeastP00924 NVNDVIAPAFVK LGANAILGVSLAASR TAGIQIVADDLTVTNPK (SEQ ID NO. 78)(SEQ ID NO. 79) (SEQ ID NO. 80) CBPY carboxypeptidase yeast P00729YDEEFASQK HFTYLR AWTDVLPWK Y (SEQ ID NO. 82) (SEQ ID NO. 83)(SEQ ID NO. 84) ADH1 alcohol yeast P00330 GVIFYESHGK SIGGEVFIDFTKVVGLSTLPEIYEK dehydrogenase 1 (SEQ ID NO. 86) (SEQ ID NO. 87)(SEQ ID NO. 88) *no analysis performed

TABLE 2  Heavy Peptide IGNIS ™ Full sequence of isotopically labeledname polypeptide + reporter peptide P I P II P III UROMDWVSVVTPARDSTIQVVENGESSQGRSGSVIDQSRLVA DWVSVVTPAR DSTIQVVENGESSQGRSGSVIDQSR LVR (SEQ ID NO. 49) (SEQ ID NO. 50) (SEQ ID NO. 51)(SEQ ID NO. 53) TRFE DGAGDVAFVKSASDLTWDNLKEGYYGYTGAFRLVALVR DGAGDVAFVKSASDLTWDNLK EGYYGYTGAFR (SEQ ID NO. 57) (SEQ ID NO. 54) (SEQ ID NO. 55)(SEQ ID NO. 56) LG3BP LADGGATNQGRSDLAVPSELALLKELSEALGQIFDSQR LADGGATNQGRSDLAVPSELALLK ELSEALGQIFDSQR LVALVR (SEQ ID NO. 58) (SEQ ID NO. 59)(SEQ ID NO. 60) (SEQ ID NO. 61) CD44FAGVFHVEKYGFIEGHVVIPRALSIGFETCRLVALVR FAGVFHVEK YGFIEGHVVIPR ALSIGFETCR(SEQ ID NO. 65) (SEQ ID NO. 62) (SEQ ID NO. 63) (SEQ ID NO. 64) CATDLVDQNIFSFYLSRVSTLPAITLKYSQAVPAVTEGPIP LVDQNIFSFYLSR VSTLPAITLKYSQAVPAVTEGPIPE EVLKLVALVR (SEQ ID NO. 66) (SEQ ID NO. 67) VLK(SEQ ID NO. 69) (SEQ ID NO. 68) KNG1TVGSDTFYSFKYFIDFVARYNSQNQSNNQFVLYRLVA TVGSDTFYSFK YFIDFVARYNSQNQSNNQFVLYR LVR (SEQ ID NO. 70) (SEQ ID NO. 71) (SEQ ID NO. 72)(SEQ ID NO. 73) ANAG LLLTSAPSLATSPAFRYDLLDLTRSDVFEAWRLVALVRLLLTSAPSLATSPAFR YDLLDLTR SDVFEAWR (SEQ ID NO. 77) (SEQ ID NO. 74)(SEQ ID NO. 75) (SEQ ID NO. 76) ENO1NVNDVIAPAFVKLGANAILGVSLAASRTAGIQIVADD NVNDVIAPAFVK LGANAILGVSLAASRTAGIQIVADDLTVTN LTVTNPKLVALVR (SEQ ID NO. 78) (SEQ ID NO. 79) PK(SEQ ID NO. 81) (SEQ ID NO. 80) CBPY YDEEFASQKHFTYLRAWTDVLPWKLVALVRYDEEFASQK HFTYLR AWTDVLPWK (SEQ ID NO. 85) (SEQ ID NO. 82)(SEQ ID NO. 83) (SEQ ID NO. 84) ADH1GVIFYESHGKSIGGEVFIDFTKVVGLSTLPEIYEKLV GVIFYESHGK SIGGEVFIDFTKVVGLSTLPEIYEK ALVR (SEQ ID NO. 86) (SEQ ID NO. 87) (SEQ ID NO. 88)(SEQ ID NO. 89)

TABLE 3  Heavy proteotypic Limit of Actual concentrationpeptide sequence Limit of detection quantification measured Proteinfrom HeavyPeptide fmol/μL Protein fmol/μL Protein fmol/μl of Proteinname MM (Da) IGNIS ™ peptide (ng/mL) peptide (ng/mL) peptide (ng/mL)Kininogen-1 71,957 TVGSDTFYSFK 0.02 1.30 0.05 3.89 197.65 14.22 (KNG1)(SEQ ID NO. 70) (SEQ ID YFIDFVAR 0.05 3.89 0.16 11.66 223.71 16.10NO. 73) (SEQ ID NO. 71) YNSQNQSNNQFVLYR 0.02 1.30 0.05 3.89 185.12 13.32(SEQ ID NO. 72)

TABLE 4 Characterization of selected human and yeast HeavyPeptide IGNIS ™Name of LVALVR universal Proteotypic peptides (PI, PH, PIII) Deltareporter (order can be changed) Reporter Mass Natural Full sequence  UP I P II P III R vs. sequence U HeavyPeptide IGNISTM UROM DWVSVVTPA*RDSTIQVV*ENGESS SGSV*IDQSR LVALVR* Tot 10 −6 DWVSVVTPA*RDSTIQVV*(SEQ ID NO. QGR (SEQ ID NO. (SEQ ID 45 ENGESSQGRSGSV*IDQS 49)(SEQ ID NO. 51) NO. 52) RLVALVR* 11 50) 10 7 (SEQ ID NO. 53) 17 TRFEDGAGDVA*FVK SA*SDLTWDNLK EGYYGYTGA*FR LV*ALV*R* 22 6 DGAGDVA*FVKSA*SDLT(SEQ ID NO. (SEQ ID NO. (SEQ ID NO. (SEQ ID 44 WDNLKEGYYGYTGA*FR 54) 55)56) NO. 52) LV*ALV*R* 11 12 12 9 (SEQ ID NO. 57) LG3BP LADGGA*TNQGRSDLAVPSELA*LLK ELSEA*LGQIFDS LVA*L*V* 51 27 11 LADGGA*TNQGRSDLAVP(SEQ ID NO. (SEQ ID NO. QR R* SELA*LLKELSEA*LGQIFD 58) 59) (SEQ ID NO.(SEQ ID SQRLVA*L*V*R* 12 14 60) NO. 52) (SEQ ID NO. 61) 15 10 CD44FA*GVFHVEK YGFIEGHVV*IPR A*LSIGFETCR LV*A*L*V* 45 33 17FA*GVFHVEKYGFIEGHVV* (SEQ ID NO. (SEQ ID NO. (SEQ ID NO. R*IPRA*LSIGFETCRLV*A* 62) 63) 64) (SEQ ID L*V*R* 10 13 11 NO. 52)(SEQ ID NO. 65) 11 CATD LV*DQNIFSF VSTLPA*ITLK YSQAVPA*VTEGPI L*V*A*L*56 40 24 LV*DQNIFSFYLSRVSTLPA* YLSR (SEQ ID NO. PEVLK V*R*ITLKYSQAVPA*VTEGPIPEV (SEQ ID NO. 67) (SEQ ID NO. (SEQ ID LKL*V*A*L*V*R*66) 11 68) NO. 52) (SEQ ID NO. 69) 14 19 12 KNG1 TV*GSDTFYS YFIDFVA*RYNSQNQSNNQFV*L LVA*LVR 47 4 −12 TV*GSDTFYSFK YFIDFVA* FK (SEQ ID NO. YR(SEQ ID R YNSQNQSNNQFV*LYR L (SEQ ID NO. 71) (SEQ ID NO. NO. 52) VA*LVR70) 10 72) 7 (SEQ ID NO. 73) 13 17 ANAG LLLTSAPSLAT YDLLDL*TR SDV*FEAWRLVALVR 41 0 −16 LLLTSAPSLATSPA*FRYDLL SPA*FR (SEQ ID NO. (SEQ ID NO.(SEQ ID DL*TRSDV*FEAWRLVALVR (SEQ ID NO. 75) 76) NO. 52) (SEQ ID NO. 77)74) 9 9 6 17 ENO1_V2 NVNDVIAPA* LGANAILGVSLAA* TAGIQIVA*DDLTV L*VA*L*VR56 20 4 NVNDVIAPA*FVKLGANAIL FVK SR TNPK (SEQ ID GVSLAA*SRTAGIQIVA*DD(SEQ ID NO. (SEQ ID NO. (SEQ ID NO. NO. 52) LTVTNPKL*VA*L*VR 78) 79) 80)9 (SEQ ID NO. 81) 13 16 18 CBPY_V2 YDEEFA*SQK HFTYL*R* A*WTDVLPWKL*VALV*R 36 13 −3 YDEEFA*SQKHFTYL*R*A* (SEQ ID NO. (SEQ ID NO.(SEQ ID NO. (SEQ ID WTDVLPWKL*VALV*R 82) 83) 84) NO. 52) (SEQ ID NO. 85)10 8 10 8 ADH1_V2 GV*IFYESHGK* SIGGEV*FIDFTK* V*VGLSTLPEIYE LVA*LVR 48 4−12 GV*IFYESHGK*SIGGEV* (SEQ ID NO. (SEQ ID NO. K* (SEQ IDFIDFTK*V*VGLSTLPEIYEK* 86) 87) (SEQ ID NO. NO. 52) LVA*LVR 12 14 88) 7(SEQ ID NO. 89) 15

TABLE 5  Name SEQ ID NO. Aqua Ultimate peptides (>97%)with labeled 13C, 15N LGANAILGVSLAASR* ENO1_A 79 TAGIQIVADDLTVTNPK*ENO1_B 80 YDEEFASQK* CBPY_A 82 HFTYLR* CBPY_B 83 GVIFYESHGK* ADH1_A 86Aqua Ultimate peptides (>97%) with labeled 13C, 15N DWVSVVTPAR* UROM_A49 DSTIQVVENGESSQGR* UROM_B 50 SGSVIDQSR* UROM_C 51 DGAGDVAFVK* TRFE_A54 SASDLTWDNLK* TRFE_B 55 EGYYGYTGAFR* TRFE_C 56 LADGGATNQGR* LG3BP_A 58SDLAVPSELALLK* LG3BP_B 59 ELSEALGQIFDSQR* LG3BP_C 60 FAGVFHVEK* CD44_A62 YGFIEGHVVIPR* CD44_B 63 ALSIGFETCR* CD44_C 64 LVDQNIFSFYLSR* CATD_A66 VSTLPAITLK* CATD_B 67 YSQAVPAVTEGPIPEVLK* CATD_C 68 TVGSDTFYSFK*KNG1_A 70 YFIDFVAR* KNG1_B 71 YNSQNQSNNQFVLYR* KNG1_C 72LLLTSAPSLATSPAFR* ANAG_A 74 YDLLDLTR* ANAG_B 75 SDVFEAWR* ANAG_C 76

The embodiments shown and described in the specification are onlyspecific embodiments of the inventor who is skilled in the art and isnot limiting in any way. Therefore, various changes, modifications, oralterations to those embodiments may be made without departing from thespirit of the invention in the scope of the following claims.

What is claimed is:
 1. A method of generating a dilution curve in asingle injection into a liquid chromatograph/mass spectrometer, themethod comprising injecting into a first liquid chromatography (LC)column coupled to a mass spectroscopy detection system a single peptidecomposition, each peptide in the composition having the identical aminoacid sequence A whereby each amino acid is present in the composition asa heavy labeled isomer resulting in the composition being a sequence Ablend of all heavy labeled isomers, each isomer in the blend of sequenceA having a mass shift and each isomer in the blend of sequence A presentin a different concentration, analyzing each peptide in the co-elutedpeptide composition by mass spectroscopy, and generating from the singlepeptide composition injection of sequence A into the LC column adilution curve from the analysis.
 2. The method of claim 1 resulting ina decreased loss of the less abundant isomer by non-specific binding toa component of the LC column due to the concentration of the mostabundant isomer being higher than the concentration of the most dilutedisomer.
 3. The method of claim 1 further comprising injecting at leastan additional sequence B and repeating the method, and comparing theresult of analysis of each of the sequence A blend and sequence B blendto compare limits of detection (LOD), and limits of quantitation (LOQ)between different column injections.
 4. The method of claim 1 furthercomprising injecting the peptide composition from claim 1 into a secondliquid chromatography (LC) column coupled to a mass spectroscopydetection system apparatus and repeating the method, and comparing theresult of analysis from each of the first column and second column tocompare limits of detection (LOD), and limits of quantitation (LOQ)between different systems.
 5. The method of claim 4 wherein the systemsare the same brand or are different brands.